Assay methods and systems

ABSTRACT

Methods, systems, kits for carrying out a wide variety of different assays that comprise providing a first reagent mixture which comprises a first reagent having a fluorescent label. A second reagent is introduced into the first reagent mixture to produce a second reagent mixture, where the second reagent reacts with the first reagent to produce a fluorescently labeled product having a substantially different charge than the first reagent. A polyion is introduced into at least one of the first and second reagent mixtures, and the fluorescent polarization in the second reagent mixture relative to the first reagent mixture is determined, this fluorescent polarization being indicative of the rate or extent of the reaction.

CROSS-REFERENCE TO RELAED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/057,812, filed Jan. 24, 2002, which is a continuation ofU.S. patent application Ser. No. 09/727,532, filed Nov. 28, 2000 (nowU.S. Pat. No. 6,436,646), which is a continuation of U.S. patentapplication Ser. No. 09/569,193, filed May 11, 2000 (now U.S. Pat. No.6,472,141), which is a continuation-in-part of U.S. patent applicationSer. No. 09/316,447, filed May 21, 1999 (now U.S. Pat. No. 6,287,774),and also claims priority to Provisional Patent Application Nos.60/139,562, filed Jun. 16, 1999 and 60/156,366, filed Sep. 28, 1999. Thedisclosure of each of these references is incorporated by reference inits entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Virtually all chemical, biological and biochemical researchdepends upon the ability of the investigator to determine the directionof her research by assaying reaction mixtures for the presence orabsence of a particular chemical species within the reaction mixture. Ina simple case, the rate or efficiency of a reaction is assayed bymeasuring the rate of production of the reaction product, or thedepletion of a reaction substrate. Similarly, interactive reactions,e.g., binding or dissociation reactions are generally assayed bymeasuring the amount of bound or free material in the resultant reactionmixture.

[0003] For certain reactions, the species of interest, or a suitablesurrogate, is readily detectable and distinguishable from the remainderof the reagents. Thus, in order to detect such species, one merely needsto look for it. Often, this is accomplished by rendering a reactionproduct optically detectable and distinguishable from the reagents byvirtue of an optical signaling element or moiety that is only present oractive on the product or the substrate. By measuring the level ofoptical signal, one can directly ascertain the amount of product orremaining substrate.

[0004] Unfortunately, many reactions of particular interest do not havethe benefit of having a readily available surrogate reagent thatproduces signal only when subjected to the reaction of interest. Forexample, many reactions that are of great interest to the biologicalresearch field do not subject their reagents to the types ofmodifications that can give rise to substantial optical propertychanges. Researchers have attempted to engineer substrates, which giverise to optical property changes. For example, typical binding reactionsbetween two molecules result in a bound complex of those molecules.However, even when one member of the binding pair is labeled, theformation of the complex does not generally give rise to an opticallydetectable difference between the complex and the labeled molecule. As aresult, most binding assays rely upon the immobilization of one memberor molecule of the binding pair. The labeled molecule is then contactedwith the immobilized molecule, and the immobilizing support is washed.Following washing, the support is then examined for the presence of thelabeled molecule, indicating binding of the labeled component to theunlabeled, immobilized component. Vast arrays of different bindingmember pairs are often prepared in order to enhance the throughput ofthe assay format. See, e.g., U.S. Pat. No. 5,143,854 to Pirrung et al.

[0005] Alternatively, in the case of nucleic acid hybridization assays,researchers have developed complementary labeling systems that takeadvantage of the proximity of bound elements to produce fluorescentsignals, either in the bound or unbound state. See, e.g., U.S. Pat. Nos.5,668,648, 5,707,804, 5,728,528, 5,853,992, and 5,869,255 to Mathies etal. for a description of FRET dyes, and Tyagi et al. Nature Biotech.14:303-8 (1996), and Tyagi et al., Nature Biotech. 16:49-53 (1998) for adescription of molecular beacons.

[0006] As noted above, binding reactions are but one category of assaysthat generally do not produce optically detectable signals. Similarly,there are a number of other assays whose reagents and/or products cannotbe readily distinguished from each other, even despite the incorporationof optically detectable elements. For example, kinase assays thatincorporate phosphate groups onto phosphorylatable substrates do notgenerally have surrogate substrates that produce a detectable signalupon completion of the phosphorylation reaction. Instead, such reactionstypically rely upon a change in the structure of the product, whichstructural change is used to separate the reactants from the product.The separated product is then detected. As should be apparent, assaysrequiring additional separation steps can be extremely time consumingand less efficient, as a result of losses during the various assaysteps.

[0007] It would generally be desirable to be able to perform theabove-described assay types without the need for solid supports,additional separation steps, or the like. The present invention meetsthese and a variety of other important needs.

SUMMARY OF THE INVENTION

[0008] The present invention provides methods, systems, kits and thelike for carrying out a wide variety of different assays. These assaystypically comprise providing a first reagent mixture which comprises afirst reagent having a fluorescent label. A second reagent is introducedinto the first reagent mixture to produce a second reagent mixture,where the second reagent reacts with the first reagent to produce afluorescently labeled product having a different charge than the firstreagent. A polyion is introduced into at least one of the first andsecond reagent mixtures, and the fluorescent polarization in the secondreagent mixture relative to the first reagent mixture is determined,this fluorescent polarization being indicative of the rate or extent ofthe reaction.

[0009] Another aspect of the present invention is a method of detectinga reaction. The method comprises providing a first reagent mixture,which contains a first reagent having a fluorescent label. A secondreagent is introduced into the first reagent mixture to produce a secondreagent mixture. The second reagent reacts with the first reagent toproduce a fluorescently labeled product having a different charge thanthe first reagent. A polyion is introduced into at least one of thefirst and second reagent mixtures and fluorescent polarization iscompared in the second reagent mixture relative to the first reagentmixture.

[0010] A further aspect of the present invention is a method ofidentifying the presence of a subsequence of nucleotides in a targetnucleic acid. The method comprises contacting the target nucleic acidsequence with a positively charged or substantially uncharged,fluorescently labeled nucleic acid analog in a first reaction mixture.The nucleic acid analog is complementary to the subsequence whereby thenucleic acid analog is capable of specifically hybridizing to thesubsequence to form a first hybrid. The first reaction mixture iscontacted with a polyion and the level of fluorescence polarization ofthe first reaction mixture in the presence of the polyion is compared tothe level of fluorescence polarization of the nucleic acid analog in theabsence of the target nucleic acid sequence. An increase in the level offluorescence polarization indicates the presence of the first hybrid.

[0011] Another aspect of the present invention is a method of detectingthe phosphorylation of a phosphorylatable compound. The method comprisesproviding the phosphorylatable compound with a fluorescent label. Thephosphorylatable compound is contacted with a kinase enzyme in thepresence of a phosphate group in a first mixture and then contacting thefirst mixture with a polyion. The level of fluorescence polarizationfrom the first mixture in the presence of the polyion is compared to thelevel of fluorescence polarization from the phosphorylatable compoundwith the fluorescent label in the absence of the kinase enzyme.

[0012] A further aspect of the present invention is a method ofdetecting the phosphorylation of a phosphorylatable compound. The methodcomprises providing the phosphorylatable compound with a fluorescentlabel. The phosphorylatable compound is contacted with a kinase enzymein the presence of a phosphate group in a first mixture. The firstmixture is contacted with a second reagent mixture comprising a proteinhaving a chelating group associated therewith, and a metal ion selectedfrom Fe³⁺, Ca²⁺, Ni²⁺ and Zn²⁺. The level of fluorescence polarizationfrom the first mixture in the presence of the second mixture is comparedto the level of fluorescence polarization from the phosphorylatablecompound with the fluorescent label in the absence of the kinase enzyme.

[0013] A further aspect of the present invention is an assay systemcomprising a fluid receptacle. The system contains a first reaction zonecontaining a first reagent mixture which comprises a first reagenthaving a fluorescent label, a second reagent that reacts with the firstreagent to produce a fluorescently labeled product having a differentcharge than the first reagent, and a polyion. The system also includes adetection zone and a detector disposed in sensory communication with thedetection zone. The detector is configured to detect the level offluorescence polarization of reagents in the detection zone.

[0014] Another aspect of the present invention is an assay systemcomprising a first channel disposed in a body structure. The firstchannel is fluidly connected to a source of a first reagent mixturewhich comprises a first reagent having a fluorescent label, a source ofa second reagent that reacts with the first reagent to produce afluorescently labeled product having a different charge than the firstreagent; and a source of a polyion. The system also includes a materialtransport system for introducing the first reagent, the second reagentand the polyion into the first channel and a detector disposed insensory communication with the first channel. The detector is configuredto detect the level of fluorescence polarization of reagents in thedetection zone.

[0015] Another aspect of the present invention is a kit. The kitincludes a volume of a first reagent which comprises a fluorescentlabel; a volume of a second reagent which reacts with the first reagentto produce a fluorescent product having a different charge from thefirst reagent; and a volume of a polyion. The kit also includesinstructions for determining the level of fluorescence polarization ofthe first reagent, mixing the first reagent, the second reagent and thepolyion in a first mixture, determining the level of fluorescencepolarization of the first mixture, and comparing the level fluorescencepolarization of the first reagent to the level of fluorescencepolarization of the first mixture.

[0016] Another aspect of the present invention is an assay system forquantifying a reaction parameter which comprises providing a firstreagent mixture. The first reagent mixture includes a first reagenthaving a fluorescent label. A second reagent is introduced into thefirst reagent mixture to produce a second reagent mixture. The secondreagent reacts with the first reagent to produce a fluorescently labeledproduct having a different charge than the first reagent. A polyion isintroduced into at least one of the first and second reagent mixtures.The system also includes a computer implemented process, comprising thesteps of determining a first level of fluorescence polarization of thefirst reagent mixture; determining a second level of fluorescencepolarization of the second reagent mixture; comparing the first andsecond levels of fluorescent polarization; and calculating the reactionparameter.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1 is a schematic illustration of one embodiment of a generalassay process performed in accordance with the present invention.

[0018]FIG. 2 is a schematic illustration of a binding assay, e.g., anucleic acid hybridization assay, performed in accordance with thepresent invention.

[0019]FIG. 3 is a schematic illustration of an enzyme assay, e.g., akinase assay, performed in accordance with the present invention.

[0020]FIG. 4 is a schematic illustration of a phosphatase assayperformed in accordance with the present invention.

[0021]FIG. 5 is a general schematic illustration of an overall systemused to carry out the assay methods of the present invention.

[0022]FIG. 6 is a schematic illustration of a multi-layered microfluidicdevice that is optionally employed as a reaction/assay receptacle in thepresent invention.

[0023]FIG. 7 is a schematic illustration of a microfluidic deviceincorporating an external sampling pipettor as a reaction/assayreceptacle in the present invention.

[0024]FIG. 8 is a schematic illustration of one example of an opticaldetection system for use with the present invention.

[0025]FIG. 9 is a flow chart of a software program or computerimplemented process carried out by an assay system in performing theassays of the present invention.

[0026]FIGS. 10A and 10B illustrate an exemplary computer system andarchitecture for use with the present invention.

[0027]FIG. 11 illustrates the interfacing of a microfluidic device withother elements of a system for controlling material movement, detectingassay results from the microfluidic device, and analyzing those results.

[0028] FIGS. 12A-E are plots of fluorescent polarization of differentfluorescent phosphorylated compounds in the presence of increasingamounts of a polycation.

[0029]FIG. 13 is a plot of fluorescent polarization of a mixture offluorescent phosphorylatable substrate and phosphorylated product, wherethe relative concentrations of substrate and product are varied in thepresence of a polycation.

[0030]FIG. 14 is a similar plot to that shown in FIG. 13, exceptutilizing a different phosphorylatable substrate and phosphorylatedproduct.

[0031]FIG. 15 is a plot of the correlation between activity of proteinkinase B (PKB) when detected using capillary electrophoreticseparation/detection (vertical axis) and fluorescent polarizationdetection (horizontal axis).

[0032]FIG. 16 is a plot of fluorescence polarization vs. reaction timefor PKA assays carried out in the presence of several differentconcentrations of ATP in the reaction mix.

[0033]FIG. 17 is a plot of initial reaction rate vs. ATP concentration,as derived from the data shown in FIG. 16.

[0034]FIG. 18 is a Lineweaver-Burke Plot derived from the assay data setforth in FIGS. 16 and 17.

[0035]FIG. 19 is the plot of phosphatase activity for the control andenzyme assay mixtures over time.

[0036]FIG. 20 is a plot of fluorescent polarization level versus timefor each of three different protease assay runs (negative control andtwo different enzyme concentrations).

[0037]FIG. 21 is a bar graph of the fluorescent polarization level of afluorescent PNA probe used to interrogate a non-complementary andcomplementary target DNA sequence, in the absence and the presence ofvarying levels of polycation.

[0038]FIG. 22 is a plot of fluorescent polarization of a mixture of atarget nucleic acid sequence and a perfectly complementary fluorescentPNA probe (upper line, diamonds) and a mixture of a target sequence thatis complementary but for a single base mismatch with a fluorescent PNAprobe (lower line, squares).

[0039]FIGS. 23A, B and C are plots of melting curves of DNA/PNA hybridswhere the target sequences and probes comprise three differentconfigurations. Shown are target sequences representing a perfect match,and two different mismatches for the target (FIGS. 23A, B and C,respectively), and three different probe lengths (9-mer, 11-mer and13-mer) as represented by the three lines in each plot.

[0040]FIG. 24 is a plot of SNP detection using the methods of thepresent invention fluorescence polarization detection and comparing thatdetection method to simple fluorescence intensity measurements.

DETAILED DESCRIPTION OF THE INVENTION I. INTRODUCTION

[0041] The present invention generally provides assay methods andsystems which are broadly useful in a variety of different contextswhere other typical assay formats cannot be used. The present methodsand systems are capable of detecting a reaction product in the presenceof the reaction substrate, despite the fact that the product is detectedby virtue of a property that it shares with the reaction substrate,e.g., a fluorescent labeling group.

[0042] In general, the methods and systems of the present inventiondistinguish reaction product from a reaction substrate by virtue of achange in the level of charge between the two as a result of thereaction. The charge on one of these reaction components, whether it islocated on the substrate or the product, is used to associate thatcomponent with a relatively large polyionic compound. The preferentialassociation of the large polyionic compound with either the substrate orthe product results in a substantial difference in the level ofpolarization of fluorescent emissions from that component when it isexcited using polarized light. Because the large compound associatespreferentially with only one of the substrate or product, as a result ofthe availability or elimination of charge due to the reaction ofinterest, that association and its consequent change in fluorescencepolarization becomes an indicator of the progress of the reaction ofinterest.

II. ASSAY METHODS

[0043] In at least one aspect, the present invention provides a methodof detecting a chemical, biochemical or biological reaction. The methodcomprises providing a first reagent mixture, which comprises a firstreagent having a fluorescent label. A second reagent is introduced intothe first reagent mixture to produce a second reagent mixture. Thesecond reagent is generally capable of reacting or otherwise interactingwith the first reagent to produce a fluorescently labeled product havinga substantially different charge than the first reagent. As used herein,the phrase “different charge” or “substantially different charge” meansthat the net charge on the product differs from that of the firstreagent by an amount sufficient to permit the differential associationof the substrate and product with a polyionic compound as describedherein. This differential charge may be a fraction of a charge, onaverage over the entire reagent molecule. However, in preferred aspects,the substrate and product typically differ by at least one charge unitat the pH at which the assay is being performed. For example, a productthat bears a net single positive or negative charge has a substantiallydifferent charge from a first reagent that has a net neutral charge, asthe phrase is used herein. Similarly, a product that bears two positivecharges has a substantially different charge from a first reagent thathas a single positive charge. Preferably, a product having asubstantially different charge than the first reagent will differ in netcharge by at least two charge units, and in certain aspects, many morethan two charge units difference, e.g., in the nucleic acid applicationsdescribed in greater detail below.

[0044] A polyion is contacted with either or both of the first andsecond reagent mixtures, depending upon the assay type that is beingperformed, and the nature of the product produced by the reaction ofinterest. Because the reaction of interest produces a product with asubstantially different charge than the substrate, that product willinteract quite differently with the polyion than will the substrate,e.g., increased or decreased interaction/association. The association orlack of association has a profound effect on the ability of the productto depolarize emitted fluorescence. Specifically, and as described ingreater detail below, the relatively small first reagent has arelatively fast rotational diffusion rate. This rotational diffusionrate is responsible for a fluorescent compound's ability to emitdepolarized fluorescence when excited with polarized light. However,association of a large polyionic compound with a small fluorescentmolecule will significantly slow the rate of rotational diffusion ofthat molecule, and reduce its ability to emit depolarized fluorescence.

[0045] The level of fluorescent polarization in the second reagentmixture is then compared to the level of fluorescent polarization fromthe first reagent mixture. By comparing these values, one can quantifythe amount of fluorescence that is emitted from material bound to thepolyion. As described in greater detail below, the assay is adjusted,e.g., by adjusting pH, ionic strength or the like, so that only one ofthe first reagent or product is capable of associating with the polyion.Thus, the change in fluorescence polarization is used to calculate aquantitative measure of the amount of product produced or first reagentconsumed, and therefore becomes a measure of the reaction.

[0046] The principles behind the use of fluorescence polarizationmeasurements as a method of measuring binding among different moleculesare relatively straight-forward. Briefly, when a fluorescent molecule isexcited with a polarized light source, the molecule will emitfluorescent light in a fixed plane, e.g., the emitted light is alsopolarized, provided that the molecule is fixed in space. However,because the molecule is typically rotating and tumbling in space, theplane in which the fluoresced light is emitted varies with the rotationof the molecule (also termed the rotational diffusion of the molecule).Restated, the emitted fluorescence is generally depolarized. The fasterthe molecule rotates in solution, the more depolarized it is.Conversely, the slower the molecule rotates in solution, the lessdepolarized, or the more polarized it is. The polarization value (P) fora given molecule is proportional to the molecule's “rotationalcorrelation time,” or the amount of time it takes the molecule to rotatethrough an angle of 57.3° (1 radian). The smaller the rotationalcorrelation time, the faster the molecule rotates, and the lesspolarization will be observed. The larger the rotational correlationtime, the slower the molecule rotates, and the more polarization will beobserved. Rotational relaxation time is related to viscosity (i),absolute temperature (T), molar volume (V), and the gas constant (R).The rotational correlation time is generally calculated according to thefollowing formula:

Rotational Correlation Time=3ηV/RT   (1)

[0047] As can be seen from the above equation, if temperature andviscosity are maintained constant, then the rotational relaxation time,and therefore, the polarization value, is directly related to themolecular volume. Accordingly, the larger the molecule, the higher itsfluorescent polarization value, and conversely, the smaller themolecule, the smaller its fluorescent polarization value.

[0048] In the performance of fluorescent binding assays, a typicallysmall, fluorescently labeled molecule, e.g., a ligand, antigen, etc.,having a relatively fast rotational correlation time, is used to bind toa much larger molecule, e.g., a receptor protein, antibody etc., whichhas a much slower rotational correlation time. The binding of the smalllabeled molecule to the larger molecule significantly increases therotational correlation time (decreases the amount of rotation) of thelabeled species, namely the labeled complex over that of the freeunbound labeled molecule. This has a corresponding effect on the levelof polarization that is detectable. Specifically, the labeled complexpresents much higher fluorescence polarization than the unbound, labeledmolecule.

[0049] Generally, the fluorescence polarization level is calculatedusing the following formula:

P=[I(∥)−I(⊥)]/[I(∥)+I(⊥)]  (2)

[0050] Where I(∥) is the fluorescence detected in the plane parallel tothe excitation light, and I(⊥) is the fluorescence detected in the planeperpendicular to the excitation light.

[0051] In performing screening assays, e.g., for potential inhibitors,enhancers, agonists or antagonists of the binding function in question,the fluorescence polarization of the reaction mixture is compared in thepresence and absence of different compounds, to determine whether thesedifferent compounds have any effect on the binding function of interest.In particular, in the presence of inhibitors of the binding function,the fluorescence polarization will decrease, as more free, labeledligand is present in the assay. Conversely, enhancers of the bindingfunction will result in an increase in the fluorescent polarization, asmore complex and less free-labeled-ligand are present in the assay.

[0052] The preferred methods of the present invention typically involvethe use of fluorescence polarization detection, e.g., detecting changesin the amount of depolarized fluorescence emitted from the reactionmixture following a given reaction. However, other fluorescent detectionschemes are also useful in the context of the present invention. Forexample, it has been discovered that in addition to altering the amountof depolarized fluorescence emitted from the reaction mixture, theassociation of the polyions in that mixture can also have an effect onthe level of overall fluorescence, or fluorescent intensity, emittedfrom the reaction mixture. Thus, in accordance with the broadest aspectsof the invention, one can merely detect a change in a variety offluorescent properties following the reaction.

[0053] As noted above, the assay methods of the present inventiontypically utilize a first reagent that includes a fluorescent labelinggroup. The nature of the first reagent generally depends upon the typeof assay that is being performed. Typically, the assays that may beperformed utilizing the present invention are myriad, including a widerange of binding or other associative assays, as well as assays ofenzymatic activity. Accordingly, the first reagents, as describedherein, generally include, e.g., one member of a specific binding pair,i.e., antibody/antigen pairs, receptor/ligand pairs, complementarynucleic acids or analogs thereof, binding proteins and their bindingsites. Alternatively, or additionally, the first reagent may comprise asubstrate which is modified by the reaction of interest, e.g., byaddition to, subtraction from or alteration of the chemical structure ofthe first reagent. Some specific examples of such substrates include,e.g., kinase substrates which include phosphorylatable moieties, e.g.,serine, threonine and tyrosine phosphorylation sites, and the like,phosphorylated substrates for phosphatase enzymes, amino or ketocontaining substrates subject to amino transferases, alcohols convertedto carboxyls (e.g., via glucose-6-phosphate dehydrogenase), as well assubstrates for: sulfatases; phosphorylases; esterases; hydrolases (e.g.,proteases); oxidases, and the like.

[0054] The first reagent may be charged, either positively ornegatively, or it may be neutral, depending upon the nature of the assaythat is to be performed. The fluorescent label on the first reagent maybe selected from any of a variety of different fluorescent labelingcompounds. Generally, such fluorescent labeling materials arecommercially available from, e.g., Molecular Probes (Eugene, Oreg.).Typically, fluorescein or rhodamine derivatives are particularly wellsuited to the assay methods described herein. These fluorescent labelsare coupled to the first reagent, e.g., covalently through well knowncoupling chemistries. For a discussion of labeling groups andchemistries, see, e.g., Published International Patent Application No.WO 98/0023 1, which is incorporated herein by reference.

[0055] Also as noted above, the second reagent generally reacts,interacts or otherwise associates or binds with the first reagent toproduce a fluorescent product that includes a substantially differentcharge than the first reagent. As with the first reagent, this secondreagent optionally comprises one member of a specific binding pair,e.g., the member that is complementary to the first reagent, providedthat the hybrid of the two members of the binding pair, or first andsecond reagents, bears a charge that is substantially different from thecharge of the first reagent member of the binding pair. In many cases,this involves a second reagent that is charged while the first reagentis neutral, or a second reagent that is highly charged as compared to afirst reagent that is only moderately charged. Alternatively, theassociation of the first and second reagents confers a conformationalchange that yields a charged product, or binds to and masks chargedresidues on the first reagent.

[0056] Because the product produced by the interaction of the first andsecond reagents has a different charge than the first reagent alone, itwill interact differently with other charged molecules. In particular,polyionic compounds that bear a substantial number of charges willgenerally interact with charged materials in a charge dependent manner.In the case of the present invention, large polyionic compounds are usedin order to “tag” the product (or in some cases, the first reagent) witha relatively large compound that will affect the product's ability toemit depolarized fluorescence.

[0057] Preferred polyions for use in the present invention includepolyamino acids, e.g., proteins, polypeptides, i.e., polylysine,polyhistidine, and polyarginine. Other polyions that are useful inaccordance with the invention include organic polyions, i.e.,polyacrylic acid, polycarboxylic acids, polyamines (e.g.,polyethylamine), polysulfonic acids (e.g., polystyrene sulfonic acid)polyphosphoric acid (e.g., polyvinylphosphoric acid), or copolymers ofany or all of these, e.g., mixed polymers of these polyamino acids, andthe like. These polyions are typically relatively large in comparison tothe first and/or second reagents, and/or the product that is being usedin the assay of interest. As such, the size of the polyion may varydepending upon the size of the first and/or second reagents and/or theproduct. Typically, the polyion will range in size from about 5 kD toabout 1000 kD, preferably, from about 10 kD to about 200 kD, and morepreferably, from about 10 kD to about 100 kD. In the case of polyaminoacids, this typically constitutes a polymer of from about 50 to about10,000 amino acid monomers in length, and preferably from about 100 toabout 1000 monomers in length.

[0058] The polyions used in accordance with the present invention aregenerally capable of interacting with the other components of thereaction mixture in a non-specific charge dependent manner. As anon-specific interaction, it will be appreciated that the polyions usedin accordance with the present invention do not require the presence ofa specific recognition site in the product (or substrate). Thisnon-specific interaction thus provides for a broader applicability forthe assays of the present invention. Also as noted, the polyioninteracts with the product (or substrate) in a charge dependent fashion.As a result, in the case of titratable polyions, this can require bufferconditions that permit the presence of the charges used in thatinteraction. Typically, polyionic materials preferably will have anisoelectric point (pI) that provides a significant level of charge atthe relevant pH level for the assay conditions. Typically, buffers inwhich the assays of the present invention are carried out will typicallybe in the physiologically relevant range, e.g., from about pH 6 to aboutpH 8, at which, the polyionic compounds will have a sufficient chargelevel to interact with charged reagents. However, as will beappreciated, one can generally adjust the level of charge, and thus thelevel of interaction between a polyion and a product (or substrate) byadjusting the buffer in which these elements are disposed, therebyeffecting the charge level of one or both of the polyion or the product.Routine reaction tuning can also be used to optimize any given assay toyield optimal reaction rates as well as interaction between thepolyionic component and the product (or substrate).

[0059] The differential interaction between the polyion and thefluorescent product, as compared to its interaction with the fluorescentfirst reagent is then used as a means for comparing the amount ofproduct produced. Specifically, a relatively small fluorescent compound,e.g., the first reagent, generally emits relatively depolarizedfluorescence when it is excited by polarized excitation light. This isgenerally due to the faster rotational diffusion or “spin” of thesesmaller compounds. Larger compounds, on the other hand have slower spinand thus are more likely to emit relatively polarized fluorescence whenexcited by a polarized excitation light source. By tagging the productwith a large “label” in the form of a polyion, one substantially altersthe product's ability to emit depolarized fluorescence. This property isthen detected and quantified as a measure of the reaction of the firstand second reagents. Typically, the detected fluorescence polarization,or P value, provides a measure of the ratio of bound label to freelabel, although assay results may also be determined as a differencebetween pre-reaction fluorescence polarization and post-reactionfluorescence polarization, with the difference being an indication ofthe reaction's rate and/or completeness.

[0060]FIG. 1 schematically and generally illustrates the assay methodsof the present invention. These illustrations are for example purposesonly and are not intended to imply limits to the present invention.Briefly, as shown in FIG. 1, a fluorescently labeled first reagent 102is provided. The first reagent has a relatively fast rotationaldiffusion rate. The first reagent 102 is contacted with a secondreagent, e.g., Enzyme I, which either mediates addition of or itselfconstitutes a charged group 104 that associates with the first reagent102 to create a charged product 106. The resulting charged producttypically has a rotational diffusion rate that is not substantiallydifferent from that of the first reagent.

[0061] The product is then contacted with a relatively large polyion108, which associates with the charged fluorescent product 106. Theresulting polyion/charged fluorescent product 110 has a substantiallyreduced rotational diffusion rate as compared to the original firstreagent. As described above, this difference in rotational diffusionrate is quantitatively measurable using, e.g., fluorescent polarizationdetection methods.

[0062] Although generally described in terms of the polyion associatingwith the product of the reaction of interest, the methods describedherein also operate in the reverse direction. Specifically, the firstreagent optionally is associated with the polyion, with all of thecharacteristics that entails. The reaction of interest then alters thecharge of the first reagent in producing a product. The product then hasa reduced or eliminated interaction with the polyion as compared to thefirst reagent. This reduced interaction then gives rise to a change inthe product's ability to emit polarized fluorescence relative to thefirst reagent. In some cases, this may require that the assay utilize aheterogeneous format, e.g., introducing the polyion after the reactionof interest is carried out, as a result of potential interfering effectsof the polyion on the reaction of interest. Methods of performing theassay methods of the invention in both homogeneous and heterogeneousformats are described in greater detail below.

[0063] The level of fluorescence polarization of the product thenprovides an indication of the amount of the fluorescent label that isbound to the polyion, e.g., as the ratio of bound to free label.Typically, fluorescence polarization data are generally reported as theratio of the difference of parallel and perpendicular fluorescenceemissions to the sum of these fluorescent emissions. Thus, the smallerthe difference between these fluorescence emissions, e.g., the moredepolarized the emissions, the smaller the polarization value.Conversely, more polarized emissions yield larger numbers. As alluded toabove, in comparing assay results, the polarization value (P) for thereaction mix is determined. The fraction of bound fluorescence, e.g.,associated with the polyion, is determined as:

F _(b)=(P−P _(f))/(P _(b) −P _(f))   (3)

[0064] where P_(b) is the P value of the bound species, and P_(f) is theP value of the free species. Thus, the polarization value can be used asan absolute quantitative measurement of the ratio of product tosubstrate, where one has determined or is already aware of the P valuefor completely bound label and completely free label. Alternatively, asnoted above, one can measure the pre-reaction and post reactionfluorescence polarization, using the difference between the two as anindication of the amount of product produced. As noted above, the assaymethods also works for the inverted assay format, e.g., where thepolyion is associated with the first reagent, but not the fluorescentproduct. In this case, the difference between the fluorescentpolarization of the first reagent and the product is determined.

[0065] The P value serves as an indicator of the reaction of interest,e.g., by indicating the amount of product produced. As will be discussedin greater detail below, once an assay reaction is quantifiable, one canuse that assay in a number of different applications, including forexample diagnostics, but particularly for screening of potentialinhibitors or enhancers of the reaction of interest. This is typicallyuseful in screening compound libraries against pharmacologicallyrelevant targets that utilize one or more of the reactions describedherein, e.g., binding, enzymatic modification and the like.

[0066] Although generally described in terms of detection of fluorescentpolarization, it will be readily appreciated that a variety of detectionschemes may be employed which detect the rate of rotation of a moleculeor the translation or lateral diffusion of a molecule that relates tothe size of the molecule. Examples of methods of detecting a molecule'srotation include, e.g., nuclear magnetic resonance spectroscopy,electron spin resonance spectroscopy, and triplet state absorbanceanisotropy. Examples of methods of detecting the translation rate ofmolecules include, e.g., fluorescent correlation spectroscopy,fluorescence recovery after photobleaching, and magnetic resonance spinexchange spectroscopies.

[0067] As noted repeatedly above, the general methods and systems of thepresent invention can be used in assaying a variety of different typesof biologically or biochemically relevant reactions, including enzymemediated reactions, binding reactions and hybridization reactions.

[0068] In the case of binding reactions, the first reagent that bears afluorescent label is contacted with a second reagent that binds to thefirst reagent to yield a fluorescently labeled product. The secondreagent typically includes a level of charge such that the productresulting from the binding of the second reagent to the first reagenthas a charge that is substantially different than the first reagentalone.

[0069] A simple example of such a binding assay is a nucleic acidhybridization assay. Specifically, in determining the presence of aparticular nucleic acid sequence or subsequence in a sample or targetnucleic acid, one often interrogates the target nucleic acid withshorter nucleic acid probes that have a nucleotide sequence that iscomplementary to, and thus is capable of hybridizing to the sequence orsubsequence of interest in the target. If the probe hybridizes to thetarget sequence, the presence of the subsequence of interest isindicated. Previously described high throughput methods have generallyrequired that at least one of the probe or the target sequence isimmobilized, e.g., on a solid support or in a particular position in anoligonucleotide array (See, e.g., U.S. Pat. Nos. 5,143,854 and5,744,305). While some solution based hybridization detection methodshave been described, these typically require specially synthesizedreagents for the sequence that is to be interrogated, e.g., includingFRET dye pairs, molecular beacons, or the like.

[0070] In the case of the present invention, the first reagent istypically a substantially uncharged or positively charged nucleic acidanalog, which bears a fluorescent label. Suitable nucleic acid analogsare generally known and include, e.g., peptide nucleic acids (PNAs),methyl phosphonate polymers and cationic nucleic acid analogs. By way ofexample, PNAs generally comprise an uncharged peptide backbone uponwhich nucleobases are disposed, as contrasted with the highly chargedglycophosphate backbones of nucleic acid molecules. PNAs are typicallypreferred due to their wide commercial availability, as well as theirexhibition of favorable hybridization properties with respect tocomplementary nucleic acid strands, e.g., higher melting points, etc.Because these nucleic acid analogs are neutral, or in some casespositively charged, they do not form a charge based association with thepolycation component of the assay, which in the case of nucleic acidassays of the invention, are positively charged polyions. For purposesof the present invention, it will be appreciated that one importantfeature of the nucleic acid analog is its inability to interact,separately, with the polyion component. Typically, this means that thenucleic acid analog will be substantially uncharged, e.g., havinginsufficient charge to interact with the polyion. Of course, in manycases, the analog will have some level of charge, e.g., associated witha fluorescent label, or will have the same type of net charge as thepolyion, e.g., either positive or negative, so as to preventinteraction. For example, in the case of nucleic acid assays, the analogcan generally be positively charged or substantially uncharged.

[0071] Because nucleic acids are highly charged species, a substantiallyuncharged or positively charged nucleic acid analog is used as the firstreagent. This permits differentiation between the free probe and theprobe that is hybridized to the target sequence by virtue of the chargeon the hybrid from the presence of the target sequence. Although thepolyion will associate with all of the target sequence, including thatwhich does not hybridize to the probe, that interaction is invisible tothe investigator, as the result of that interaction not bearing afluorescent label. This nucleic acid hybridization assay isschematically illustrated in FIG. 2.

[0072] As shown, a target nucleic acid 202 (schematically illustrated)is interrogated with a fluorescent probe 204 (the fluorescent label isindicated as an *), which typically comprises a positively charged orsubstantially uncharged nucleic acid analog, e.g., a PNA probe. Theprobe is selected to be complementary to a particular nucleotidesequence, e.g., the sequence of interest, such that the probe willselectively hybridize to that sequence if it is present in the targetnucleic acid 202. In its individual form, the probe will have arelatively high rate of rotational diffusion due to its small size, asschematically illustrated by arrow 206, thereby emitting more highlydepolarized fluorescence.

[0073] The reaction illustrated in panel I illustrates the case wherethe target sequence 202 contains the sequence of interest, so that theprobe 204 will hybridize to the target sequence 202 to form a firsthybrid 208. Due to the larger size of the hybrid relative to that of theprobe, this hybridization reaction will result in a reduction in therotational rate of diffusion of the fluorescently labeled compound (inthis case, the hybrid), as indicated by arrow 210. However, due to theflexible nature of nucleic acids, as well as the only incrementalincrease in size of the hybrid over that of the target, this reductionmay not be substantial, and may not be easily detectable. In accordancewith the methods of the present invention, however, this signal (P) iseffectively amplified by adding a polyionic compound 212 to the hybrid.Specifically, nucleic acids, i.e., the target sequence, are highlycharged species due to their negatively charged phosphate/sugarbackbones. Thus, this charge exists even when the nucleic acid exists indouble stranded form.

[0074] When a polyionic compound, e.g., polycation 212, i.e.,polylysine, is added to the hybrid 208, it associates with the hybrid208, in an associative complex 214, thereby substantially decreasing therotational diffusion of the overall complex 214, as schematicallyillustrated by arrow 222. This difference is more readily detected.

[0075] In contrast, the reaction illustrated in panel II illustrates theinstance where the target sequence 202 does not include the sequence ofinterest that is complementary to the sequence of the fluorescent probe204. As such, the probe and target sequence are unable to hybridize, andthe rotational diffusion of the fluorescent component (the unhybridizedprobe) remains unchanged as illustrated by arrow 216. Further, when thepolycationic polyion is added to the reaction, it will again associatewith the highly charged target sequence as an associative complex 218.However, the polycation will not associate with the fluorescent probe204 due to the probe's uncharged or like-charged character. As such, therotational diffusion of the fluorescent compound (again the unhybridizedprobe 204) will remain unchanged, as illustrated by arrow 220. As aresult, in the case where hybridization occurs, i.e., the sequence ofinterest is present in the target, the fluorescence emissions from thereaction, when excited by polarized excitation light, is substantiallypolarized as compared to that of the unhybridized probe. Conversely,where no hybridization occurs, i.e., the sequence of interest is notpresent, there is no change in the level of fluorescence polarization.Accordingly, a change in fluorescence polarization becomes an indicatorof the presence of the sequence of interest.

[0076] The methods and systems of the present invention are also usefulin carrying out a variety of other binding assays, where the resultingcomplex has a substantially different charge from the charge of afluorescently labeled member of the binding pair. For example, in areceptor binding assay where a neutral, fluorescent ligand is bound tothe charged, unlabeled receptor, the methods of the present inventionserve to amplify a fluorescence polarization signal from the complex byassociating a large polyionic compound with that complex. Specifically,while the complex will, by itself, give a fluorescence polarizationresponse, as described herein, the complex with the associated polyioniccompound will be substantially greater. As will be appreciated, a widevariety of binding assays may be carried out in accordance with thepresent invention. Even a greater number of assays may be readilyconfigured to function in accordance with these methods, e.g., where thebound complex has a substantially different charge than a fluorescentlylabeled free member of the ultimate complex.

[0077] The methods and systems of the present invention also findparticular usefulness in assaying for enzymatic activity where thatactivity produces a product that has a substantially different chargethan the substrate upon which the enzyme acted. One example of a classof enzyme assays that is suited for the methods and systems of thepresent invention are those that add or remove phosphate groups to orfrom appropriate substrates, e.g. kinase and phosphatase assays.Interest in these activities is substantial due to their roles inmediating a wide variety of biologically relevant response reactions invivo. In particular, kinase and phosphatase reactions are oftenprecursor, or intermediate signaling events in complex cellularbehaviors such as survival and proliferation. As such, their activitiesbecome of particular interest in addressing diseases where thesebehaviors are malfunctioning, e.g., cancer, and the like.

[0078] As noted above, the present invention is particularly useful inassaying for the activity of kinase enzymes. Kinase enzymes typicallyfunction by adding a phosphate group to a phosphorylatable substrate,e.g., protein, peptide, nucleoside, carbohydrate, etc. As phosphategroups are highly charged, their addition to a particular substratetypically imparts a substantial change in charge of the product over thesubstrate. As with the assays described above, this change in charge inthe product over that of the substrate can be exploited by adding apolyionic compound that imparts a significant difference in thefluorescence polarization of the product over the substrate.

[0079] Briefly, a phosphorylatable substrate is provided with afluorescent labeling group, as described above. The phosphorylatablesubstrate may be neutral or it may be charged. Preferred substrates areneutral under the relevant assay conditions. A variety ofphosphorylatable substrates are commercially available. For example,rhodamine labeled substrate for protein kinase A (PKA) is generallycommercially available from Promega Inc., while other fluorescentphosphorylatable substrates may be obtained from Research Genetics, Inc.

[0080] Because the fluorescent phosphorylatable substrate is typicallyrelatively small, e.g., less than about 2 kD, it has a relatively highrate of rotational diffusion and thereby emits depolarized fluorescencewhen excited with polarized light. When contacted with a kinase enzymein the presence of a phosphate donor, e.g., ATP, the substrate isphosphorylated, imparting two additional negative charges for eachphosphate incorporated. This net −2 charge, particularly in the case ofthe previously uncharged substrate, provides a basis for interaction ofthe product with a polyionic compound, e.g., a polycation such aspolylysine or polyhistidine. Once the polyion associates with thephosphorylated substrate, it significantly slows the rate of rotationaldiffusion, and thereby reduces the rate of fluorescence depolarization,e.g., increases the fluorescence polarization value. This change inpolarization is then detected and used to quantify the kinase reaction.

[0081] A schematic illustration of this reaction is shown in FIG. 3. Asshown, the fluorescently labeled phosphorylatable substrate 302 iscontacted with a kinase enzyme 306, in the presence of phosphate 304,e.g., in the form of ATP. The reaction yields the phosphorylated product308. Both the fluorescent substrate 302 and the phosphorylatedfluorescent product 308 have relatively high rates of rotationaldiffusion due to their small size. The fluorescent phosphorylatedproduct is then contacted with a polycation. Preferred polycationsinclude polyamino acids such as polylysine, polyhistidine or the like,with polyhistidine being most preferred. The polycation then associateswith the negatively charged phosphorylated fluorescent product, therebydrastically affecting its size and rotational diffusion rate, which isthen detected as described repeatedly herein. As will be appreciated,the polyionic component may alternatively comprise a large molecule,e.g., a protein or the like, that has associated therewith multivalentmetal cations selected from, e g., Fe³⁺, Ca²⁺, Ni²⁺ and Zn^(2°).Examples of such molecules include metal chelating proteins that chelatethese ions, or the like. Specifically, these metal ions have relativelyhigh affinity for oxygen, nitrogen and sulfur groups. As a result, theycan impart a significant binding affinity to a large molecule (as apolyion) towards, e.g., phosphate groups in nucleic acids orphosphorylated substrates and the like, as well as other groups bearingoxygen, nitrogen or sulfur groups, giving rise to the interaction thatis used to significantly slow the rotational diffusion rate of afluorescent species, as described herein.

[0082] The present invention is also well suited to assay the reversereaction. Specifically, the phosphatase reaction, which removes aphosphate group from a phosphorylated substrate. This reaction followssubstantially the reverse path of that shown in FIG. 3, and isschematically illustrated in FIG. 4. Briefly, the fluorescentphosphorylated compound 308, which in this instance is the substrate, iscontacted with the polycationic compound 310 to yield the associativecomplex 312 where the polycation associates with the charges imparted bythe phosphate group. As noted above with reference to FIG. 3, thiscomplex has a slow rate of rotational diffusion. When this complex isacted on by a phosphatase enzyme 414 it results in cleavage of thecharged phosphate group and its associated polycation 404 from thefluorescent component 302, which in this instance is the product. Whenfree of the large polycationic compound, the fluorescent product has agreatly increased rate of rotational diffusion, e.g., emittingdepolarized fluorescence. Again, this change in fluorescencepolarization is detected as described herein. As noted above, in somecases, the assay may preferably be performed in a heterogeneous format,e.g., where the polyionic component is added after the reaction ofinterest, in order to avoid any adverse effects of the presence of thepolyion on the reaction.

[0083] While the ability to perform a variety of assays is itselfuseful, the specific applications to which these assays are puttypically provides the greatest value. Of particular interest is theability to test the effects of potential pharmaceutical candidatecompounds on the various activities described above. Specifically, inpharmaceutical discovery processes, large libraries of chemicalcompounds are generally screened against pharmacologically relevanttargets. These targets may include receptors, enzymes, transporters, andthe like. A variety of screening assays and systems have been described.See, e.g., Published International Patent Application No. WO 98/00231,which is incorporated herein by reference.

[0084] In brief, a particular reaction that is biologically orbiochemically relevant is carried out in the presence and absence of acompound that is to be screened, and the effect of the compound isdetermined. Specifically, if the reaction is slowed or blocked by thepresence of the test compound, then the compound is identified as aninhibitor of the reaction. Conversely where the reaction proceeds morerapidly or to a greater extent in the presence of the test compound,then the compound is identified as an enhancer of the reaction. Thesescreening assays are then performed for a large number of differentcompounds, either serially or in parallel, in order to expedite thediscovery of potential effectors of the reaction of interest.

III. ASSAY SYSTEMS

[0085] The present invention also provides assay systems that are usedin carrying out the above-described methods. Typically, the assaysystems described herein comprise a fluid receptacle into which thereagents are placed for performing the assay. The fluid receptacletypically comprises a first reaction zone having disposed therein afirst reagent mixture which comprises a first reagent having afluorescent label, a second reagent that reacts with the first reagentto produce a fluorescently labeled product having a substantiallydifferent charge than the first reagent and a polyionic compound.

[0086]FIG. 5 schematically illustrates an overall assay system for usein practicing the present invention. Briefly, the overall system 500includes a reaction receptacle 502, as described above. A detector ordetection system 504 is disposed adjacent to the receptacle and withinsensory communication of the receptacle. The phrase “within sensorycommunication” generally refers to the detector that is positionedrelative to the receptacle so as to be able to receive a particularsignal from that receptacle. In the case of optical detectors, e.g.,fluorescence or fluorescence polarization detectors, sensorycommunication typically means that the detector is disposed sufficientlyproximal to the receptacle that optical, e.g., fluorescent signals aretransmitted to the detector for adequate detection of those signals.Typically this employs a lens, optical train or other detection element,e.g., a CCD, that is focused upon a relevant portion of the receptacleto efficiently gather and record these optical signals.

[0087] Detector 504 is typically connected to an appropriate datastorage and/or analysis unit, e.g., a computer or other processor, whichis generally capable of storing, analyzing and displaying the obtaineddata from the receptacle in a user comprehendible fashion, e.g., display508. In certain embodiments, e.g., those employing microfluidicreceptacles, the computer 506 is optionally connected to an appropriatecontroller unit 510, which controls the movement of fluid materialswithin the channels of the microfluidic device receptacle, and/orcontrols the relative position of the receptacle 502 and detector 504,e.g., via an x-y-z translation stage.

[0088] The receptacle also typically includes a detection zone as wellas a detector disposed in sensory communication with the detection zone.The detector used in accordance with the present invention typically isconfigured to detect a level of fluorescence polarization of reagents inthe detection zone.

[0089] As used herein, the receptacle may take on a variety of forms.For example, the receptacle may be a simple reaction vessel, well, tube,cuvette, or the like. Alternatively, the receptacle may comprise acapillary or channel either alone or in the context of an integratedfluidic system that includes one or more fluidic channels, chambers orthe like.

[0090] In the case of a simple reaction vessel, well, tube, cuvette orthe like, the reaction zone and the detection zone typically refer tothe same fluid containing portion of the receptacle. For example, withinthe fluid containing portion of a cuvette, reagents are mixed, reactedand subsequently detected. Typically, in order to expedite the processof performing assays, e.g., screening assays, multiplexed receptaclesmay be used. Examples of such receptacles include, e.g., multiwellplates, i.e., 96-well, 384-well or 1536-well plates.

[0091] For capillary or channel based aspects, the reaction zone and thedetection zone may comprise the same fluid-containing portion of thereceptacle. However, in many aspects, the reaction zone and thedetection zone are separate fluid containing portions of the receptacle.Specifically, reagents may be mixed and reacted in one portion of thereceptacle, and subsequently moved to a separate detection zonewhereupon the reaction products, etc. are detected.

[0092] In particularly preferred aspects, the receptacle comprises amicrofluidic device. As used herein, the term “microfluidic device”refers to a device or body structure which includes and/or contains atleast one fluidic component, e.g., a channel, chamber, well or the like,which has at least one cross sectional dimension that is between about0.1 and about 500 μm, with these channels and/or chambers often havingat least one cross-sectional dimension between about 0.1 μm and 200 μm,in some cases between about 0.1 μm and 100 μm, and often between about0.1 μm and 20 μm. Such cross-sectional dimensions include, e.g., width,depth, height, diameter or the like. Typically, structures having thesedimensions are also described as being “microscale.” Microfluidicdevices in accordance with the present invention, typically include atleast one, and preferably more than one channel and/or chamber disposedwithin a single body structure. Such channels/chambers may be separateand discrete, or alternatively, they may be fluidly connected. Suchfluid connections may be provided by channels, channel intersections,valves and the like. Channel intersections may exist in a number offormats, including cross intersections, “T” intersections, or any numberof other structures whereby two channels are in fluid communication.

[0093] Because of their controllability, microfluidic device embodimentsof the present invention are particularly useful in carrying outheterogeneous forms of the assays described herein. In particular,reactions are performed in a first region of the microscale channelnetwork. The products of the reaction are then moved to a differentportion of the channel network, or additional components are broughtinto the original portion of the channel network to mix with theproducts of the reaction. For example, the polyionic component of theassay methods described herein can be added after the reaction ofinterest to ensure that it does not interfere with the reaction.Microfluidic systems provide the ability to precisely move the variousreagents through the various channels of the device, permitting theiraccurate measurement and timely addition. By way of a simple example, aphosphatase reaction may be carried out on a phosphorylated substrate ina first channel region of a microfluidic device, yielding a phosphategroup, e.g., ATP, and the unphosphorylated product, as well as unreactedsubstrate. The mixture is then mixed with the polyionic component, e.g.,polyhistidine, either by moving the reaction mixture to a separatechannel containing the polyion or by introducing the polyion into thereaction mixture in the original channel segment. The resulting mixtureis then moved past a detection point where the fluorescence polarizationis measured.

[0094] The body structure of the microfluidic devices described hereintypically comprises an aggregation of two or more separate componentswhich when appropriately mated or joined together, form the microfluidicdevice of the invention, e.g., containing the channels and/or chambersdescribed herein. Typically, the microfluidic devices described hereinare fabricated as an aggregate of substrate layers. In particular, suchpreferred devices comprise a top portion, a bottom portion, and aninterior portion, wherein the interior portion substantially defines thechannels and chambers of the device.

[0095]FIG. 6 illustrates a two-layer body structure 610, for amicrofluidic device. In preferred aspects, the bottom portion of thedevice 612 comprises a solid substrate that is substantially planar instructure, and which has at least one substantially flat upper surface614. A variety of substrate materials may be employed as the bottomportion. Typically, because the devices are microfabricated, substratematerials will be selected based upon their compatibility with knownmicrofabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, injection molding,embossing, and other techniques. The substrate materials are alsogenerally selected for their compatibility with the full range ofconditions to which the microfluidic devices may be exposed, includingextremes of pH, temperature, salt concentration, and application ofelectric fields. Accordingly, in some preferred aspects, the substratematerial may include materials normally associated with thesemiconductor industry in which such microfabrication techniques areregularly employed, including, e.g., silica based substrates, such asglass, quartz, silicon or polysilicon, as well as other substratematerials, such as gallium arsenide and the like. In the case ofsemiconductive materials, it will often be desirable to provide aninsulating coating or layer, e.g., silicon oxide, over the substratematerial, and particularly in those applications where electric fieldsare to be applied to the device or its contents.

[0096] In additional preferred aspects, the substrate materials willcomprise polymeric materials, e.g., plastics, such aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, polystyrene, polymethylpentene, polypropylene,polyethylene, polyvinylidine fluoride, ABS(acrylonitrile-butadiene-styrene copolymer), and the like. Suchpolymeric substrates are readily manufactured using availablemicrofabrication techniques, as described above, or from microfabricatedmasters, using well known molding techniques, such as injection molding,embossing or stamping or the like. Such polymeric substrate materialsare preferred for their ease of manufacture, low cost and disposability,as well as their general inertness to most extreme reaction conditions.Again, these polymeric materials may include treated surfaces, e.g.,derivatized or coated surfaces, to enhance their utility in themicrofluidic system, e.g., provide enhanced fluid direction, e.g., asdescribed in U.S. Pat. No. 5,885,470, which is incorporated herein byreference in its entirety for all purposes.

[0097] The channels and/or chambers of the microfluidic devices aretypically fabricated into the upper surface of the bottom substrate orportion 612 (although they are optionally fabricated into either or bothof the upper surface of the bottom substrate or the lower surface of theupper substrate) as microscale grooves or indentations 616, using theabove described microfabrication techniques. The top portion orsubstrate 618 also comprises a first planar surface 620, and a secondsurface 622 opposite the first planar surface 620. In the microfluidicdevices prepared in accordance with the methods described herein, thetop portion also includes a plurality of apertures, holes or ports 624disposed therethrough, e.g., from the first planar surface 620 to thesecond surface 622 opposite the first planar surface.

[0098] The first planar surface 620 of the top substrate 618 is thenmated, e.g., placed into contact with, and bonded to the planar surface614 of the bottom substrate 612, covering and sealing the grooves and/orindentations 616 in the surface of the bottom substrate, to form thechannels and/or chambers (i.e., the interior portion) of the device atthe interface of these two components. The holes 624 in the top portionof the device are oriented such that they are in communication with atleast one of the channels and/or chambers formed in the interior portionof the device from the grooves or indentations in the bottom substrate.In the completed device, these holes function as reservoirs forfacilitating fluid or material introduction into the channels orchambers of the interior portion of the device, as well as providingports at which electrodes may be placed into contact with fluids withinthe device, allowing application of electric fields along the channelsof the device to control and direct fluid transport within the device.

[0099] In many embodiments, the microfluidic devices will include anoptical detection window disposed across one or more channels and/orchambers of the device. Optical detection windows are typicallytransparent such that they are capable of transmitting an optical signalfrom the channel/chamber over which they are disposed. Optical detectionwindows may merely be a region of a transparent cover layer, e.g., wherethe cover layer is glass or quartz, or a transparent polymer material,e.g., PMMA, polycarbonate, etc. Alternatively, where opaque substratesare used in manufacturing the devices, transparent detection windowsfabricated from the above materials may be separately manufactured intothe device.

[0100] As described in greater detail below, these devices may be usedin a variety of applications, including, e.g., the performance of highthroughput screening assays in drug discovery, immunoassays,diagnostics, genetic analysis, and the like. As such, the devicesdescribed herein, will often include multiple sample introduction portsor reservoirs, for the parallel or serial introduction and analysis ofmultiple samples. Alternatively, these devices may be coupled to asample introduction port, e.g., a pipettor, which serially introducesmultiple samples into the device for analysis. Examples of such sampleintroduction systems are described in e.g., U.S. Pat. No. 5,779,868 andpublished International Patent Application Nos. WO 98/00705 and WO98/00231, each of which is incorporated herein by reference in itsentirety for all purposes. A schematic illustration of a microfluidicdevice incorporating an external sample pipettor is illustrated in FIG.7, described below.

[0101] In the case of some substrates, e.g., glass, quartz, or silica,it is sometimes desirable to include a coating material in the channelsof the microfluidic device. This is primarily to reduce the level ofinteraction between the polyion component and the charged surface of thesubstrate. Any of a variety of known coating materials are useful inthis regard, including polymer coatings typically used inelectrophoretic applications, e.g., linear polyacrylamides, e.g.,polydimethylacrylamides (PDMA), and the like (see, e.g., U.S. Pat. Nos.5,948,227, 5,567,292, and 5,264,101, each of which is incorporated byreference). Such polymers may be silica adsorbing, or may be covalentlyattached to the surface of the substrates, e.g., through the inclusionof an epoxide group on the polymer chain (see, e.g., Chiari et al., HPCEConference, March, 2000), in order to mask surface charges on thesubstrate which may interact with the polyionic species in the reactionmixture.

[0102] Briefly, a microfluidic device 700, e.g., similar to thatdescribed with reference to FIG. 6, is provided having a body structure702 which includes a network of internal channels 704 that are connectedto a series of reservoirs 706 disposed in the body structure 702. Thevarious reservoirs are used to introduce various reagents into thechannels 704 of the device. A capillary element 708 is coupled to thebody structrure 702, such that the channel 710 that is disposed withinand runs the length of the capillary element 708 is fluidly connected tothe channel network 704 in the body structure. This capillary element708 is then used to draw up a variety of different sample or testmaterials, in series, for analysis within the device.

[0103] As described above, the methods and systems of the presentinvention typically rely upon a change in the level of fluorescencepolarization of the reaction mixture as a result of the reaction ofinterest. As such, an appropriate detection system is typically utilizedto differentiate polarized from depolarized emitted fluorescence.Generally speaking, such a detection system typically separately detectsfluorescent emissions that are emitted in the same plane of thepolarized excitation light, and fluorescent emissions emitted in a planeother than the plane of the excitation light.

[0104] One example of a detection system is shown in FIG. 8. As shown,the fluorescence polarization detector includes a light source 804,which generates light at an appropriate excitation wavelength for thefluorescent compounds that are present in the assay system. Typically,coherent light sources, such as lasers, laser diodes, and the like arepreferred because of the highly polarized nature of the light producedthereby. The excitation light is directed through an optional polarizingfilter 806, which passes only light in one plane, e.g., polarized light.The polarized excitation light is then directed through an opticaltrain, e.g., dichroic mirror 810 and microscope objective 812 (andoptionally, reference beam splitter 808), which focuses the polarizedlight onto the sample receptacle (illustrated as a channel inmicrofluidic device 802), in which the sample to be assayed is disposed.

[0105] Fluorescence emitted from the sample is then collected, e.g.,through the objective 812, and directed back through dichroic mirror810, which passes the emitted fluorescence and reflects the reflectedexcitation light, thereby separating the two. The emitted fluorescenceis then directed through a beam splitter 814 where one portion of thefluorescence is directed through an filter 816 that filters outfluorescence that is in the plane that is parallel to the plane of theexcitation light and directs the perpendicular fluorescence onto a firstlight detector 818. The other portion of the fluorescence is passedthrough a filter 820 that filters out the fluorescence that isperpendicular to the plane of the excitation light, directing theparallel fluorescence onto a second light detector 822. In alternativeaspects, beam splitter 814 is substituted with a polarizing beamsplitter, e.g., a Glan prizm, obviating the need for filters 816 and820. These detectors 818 and 822 are then typically coupled to anappropriate recorder or processor (not shown in FIG. 8) where the lightsignal is recorded and or processed as set out in greater detail below.Photomultiplier tubes (PMTs), are generally preferred as light detectorsfor the quantification of the light levels, but other light detectorsare optionally used, such as photodiodes, or the like.

[0106] The detector is typically coupled to a computer or otherprocessor, which receives the data from the light detectors, andincludes appropriate programming to compare the values from eachdetector to determine the amount of polarization from the sample. Inparticular, the computer typically includes software programming whichreceives as input the fluorescent intensities from each of the differentdetectors, e.g., for parallel and perpendicular fluorescence. Thefluorescence intensity is then compared for each of the detectors toyield a fluorescence polarization value. One example of such acomparison is given by the equation:

P=[I(∥)−I(⊥)]/[I(∥)+I(⊥)]C   (4)

[0107] as shown above, except including a correction factor (C), whichcorrects for polarization bias of the detecting instrument. The computerdetermines the fluorescence polarization value for the reaction ofinterest. From that polarization value and based upon the polarizationvalues for free and bound fluorescence, the computer calculates theratio of bound to free fluorescence. Alternatively, the polarizationvalues pre and post reaction are compared and a polarization difference(ΔP) is determined. The calculated polarization differences may then beused as absolute values, e.g., to identify potential effectors of aparticular reaction, or they may be compared to polarization differencesobtained in the presence of known inhibitors or enhancers of thereaction of interest, in order to quantify the level of inhibition orenhancement of the reaction of interest by a particular compound.

[0108]FIG. 9 illustrates a flow-chart for the processes carried out bythe computer using the above-described software programming. As shown,the programmed process begins at step 902 where the computer receivesthe fluorescence intensity data for the unreacted reagents in thereaction zone (e.g. in receptacle 502 of FIG. 5) from the two detectors,e.g., detectors 818 and 820 of FIG. 8. The fluorescence polarizationvalue (P) is then calculated in step 904, e.g., according to theequations described herein. At step 906, the computer receivesfluorescence intensity data for the reacted reagents from the twodetectors. Again, at step 908, the P value is calculated for the reactedreagents. At step 910, the P values for the reacted and unreactedreagents are compared, e.g., one is subtracted from the other to yield aΔP value for the reaction. At this point, the ΔP value may be displayedas a measure of the reaction, e.g., its rate or completeness.Optionally, however, the ΔP value may be compared to a standard ΔPvalue, i.e., from a reaction having a known rate, level of inhibition orenhancement, e.g., at step 912. Through this comparison, the computermay then interpolate or extrapolate a quantitative measure of thereaction, its level of inhibition or enhancement which quantitativemeasurement may then be displayed to the investigator, e.g., at step914. As noted above, the computer may optionally include a determinedpolarization value for completely free and completely boundfluorescence. In that case, determination of fluorescence differences isnot necessary, thus permitting the omission of several steps of theprogram. In that case, the computer receives the fluorescence data fromthe detector for the reacted mixture. The computer then merelycalculates the P value for the reaction mixture and determines the ratioof bound fluorescence to free fluorescence (e.g., in accordance withequation (3), supra). The ratio is then used to quantitate the reaction.

[0109] In the case of high-throughput screening assay systems, thecomputer software optionally instructs the correlation of a particularscreened result to a particular sample or sample acquisition location.This permits the investigator to identify the particular reagentsemployed in any one assay.

[0110]FIG. 10 schematically illustrates a computer and architecturetypically used in accordance with the present invention. In particular,FIG. 10A illustrates an example of a computer system that may be used toexecute software for use in practicing the methods of the invention orin conjunction with the devices and/or systems of the invention.Computer system 1000 typically includes a display 1002, screen 1004,cabinet 1006, keyboard 1008, and mouse 1010. Mouse 1010 may have one ormore buttons for interacting with a graphic user interface (GUI).Cabinet 1006 typically houses a CD-ROM drive 1012, system memory and ahard drive (see FIG. 10B) which may be utilized to store and retrievesoftware programs incorporating computer code that implements themethods of the invention and/or controls the operation of the devicesand systems of the invention, data for use with the invention, and thelike. Although CD-ROM 1014 is shown as an exemplary computer readablestorage medium, other computer readable storage media, including floppydisk, tape, flash memory, system memory, and hard drive(s) may be used.Additionally, a data signal embodied in a carrier wave (e.g., in anetwork, e.g., internet, intranet, and the like) may be the computerreadable storage medium.

[0111]FIG. 10B schematically illustrates a block diagram of the computersystem 1000, described above. As in FIG. 10A, computer system 1000includes monitor or display 1002, keyboard 1008, and mouse 1010.Computer system 1000 also typically includes subsystems such as acentral processor 1016, system memory 1018, fixed storage 1020 (e.g.,hard drive) removable storage 1022 (e.g., CD-ROM drive) display adapter1024, sound card 1026, speakers 1028 and network interface 1030. Othercomputer systems available for use with the invention may include feweror additional subsystems. For example, another computer systemoptionally includes more than one processor 1014.

[0112] The system bus architecture of computer system 1000 isillustrated by arrows 1032. However, these arrows are illustrative ofany interconnection scheme serving to link the subsystems. For example,a local bus could be utilized to connect the central processor to thesystem memory and display adapter. Computer system 1000 shown in FIG.10A is but an example of a computer system suitable for use with theinvention. Other computer architectures having different configurationsof subsystems may also be utilized, including embedded systems, such ason-board processors on the controller detector instrumentation, and“internet appliance” architectures, where the system is connected to themain processor via an internet hook-up.

[0113] The computer system typically includes appropriate software forreceiving user instructions, either in the form of user input into setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the optional materialtransport system, and/or for controlling, manipulating, storing etc.,the data received from the detection system. In particular, the computertypically receives the data from the detector, interprets the data, andeither provides it in one or more user understood or convenient formats,e.g., plots of raw data, calculated dose response curves, enzymekinetics constants, and the like, or uses the data to initiate furthercontroller instructions in accordance with the programming, e.g.,controlling flow rates, applied temperatures, reagent concentrations,etc.

[0114] As described above, the present invention is optionally carriedout in a microfluidic device or system. As such, it is generallydesirable to provide a means or system for moving materials through,between and among the various channels, chambers and zones that arecontained in such devices. A variety of material transport methods areoptionally used in accordance with such microfluidic devices. Forexample, in one preferred aspect material movement through the channelsof a device is caused by the application of pressure differentialsacross the channels through which material flow is desired. This may beaccomplished by applying a positive pressure to one end of a channel ora negative pressure to the other end. In complex channel networks,controlled flow rates in all of the various interconnected channels maybe controlled by the inclusion of valves, and the like within the devicestructure, e.g., to stop and start flow through a given channel.Alternatively, channel resistances may be adjusted to dictate the rate,timing and/or volume of material movement through different channels,even under a single applied pressure differential, e.g., a vacuumapplied at a single channel port. Examples of such channel networks areillustrated in e.g., U.S. patent application Ser. No. 09/238,467, filedJan. 28, 1999 and U.S. Pat. Nos. 6,500,323 and 6,150,119, all of whichare hereby incorporated herein by reference in their entirety for allpurposes.

[0115] Alternately, for microfluidic applications of the presentinvention, controlled electrokinetic transport systems may be used. Thistype of electrokinetic transport is described in detail in U.S. Pat. No.5,858,195, to Ramsey, which is incorporated herein by reference for allpurposes. Such electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. Other electrokinetic material direction and transportsystems rely upon the electroosmotic flow of fluid and material within achannel or chamber structure which results from the application of anelectric field across such structures. In brief, when a fluid is placedinto a channel which has a surface bearing charged functional groups,e.g., hydroxyl groups in etched glass channels or glassmicrocapillaries, those groups can ionize. In the case of hydroxylfunctional groups, this ionization, e.g., at neutral pH, results in therelease of protons from the surface and into the fluid, creating aconcentration of protons at near the fluid/surface interface, or apositively charged sheath surrounding the bulk fluid in the channel.Application of a voltage gradient across the length of the channel, willcause the proton sheath to move in the direction of the voltage drop,i.e., toward the negative electrode.

[0116] “Controlled electrokinetic material transport and direction,” asused herein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. In particular, the preferred microfluidic devicesand systems described herein, include a body structure which includes atleast two intersecting channels or fluid conduits, e.g., interconnected,enclosed chambers, which channels include at least three unintersectedtermini. The intersection of two channels refers to a point at which twoor more channels are in fluid communication with each other, andencompasses “T” intersections, cross intersections, “wagon wheel”intersections of multiple channels, or any other channel geometry wheretwo or more channels are in such fluid communication. An unintersectedterminus of a channel is a point at which a channel terminates not as aresult of that channel's intersection with another channel, e.g., a “T”intersection. In preferred aspects, the devices will include at leastthree intersecting channels having at least four unintersected termini.In a basic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic material flow of this materialacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage). However, this type of materialflow through the intersection would result in a substantial amount ofdiffusion at the intersection, resulting from both the natural diffusiveproperties of the material being transported in the medium used, as wellas convective effects at the intersection.

[0117] In controlled electrokinetic material transport, the materialbeing transported across the intersection is constrained by low levelflow from the side channels, e.g., the top and bottom channels. This isaccomplished by applying a slight voltage gradient along the path ofmaterial flow, e.g., from the top or bottom termini of the verticalchannel, toward the right terminus. The result is a “pinching” of thematerial flow at the intersection, which prevents the diffusion of thematerial into the vertical channel. The pinched volume of material atthe intersection may then be injected into the vertical channel byapplying a voltage gradient across the length of the vertical channel,i.e., from the top terminus to the bottom terminus. In order to avoidany bleeding over of material from the horizontal channel during thisinjection, a low level of flow is directed back into the side channels,resulting in a “pull back” of the material from the intersection.

[0118] In addition to pinched injection schemes, controlledelectrokinetic material transport is readily utilized to create virtualvalves which include no mechanical or moving parts. Specifically, withreference to the cross intersection described above, flow of materialfrom one channel segment to another, e.g., the left arm to the right armof the horizontal channel, can be efficiently regulated, stopped andreinitiated, by a controlled flow from the vertical channel, e.g., fromthe bottom arm to the top arm of the vertical channel. Specifically, inthe ‘off’ mode, the material is transported from the left arm, throughthe intersection and into the top arm by applying a voltage gradientacross the left and top termini. A constraining flow is directed fromthe bottom arm to the top arm by applying a similar voltage gradientalong this path (from the bottom terminus to the top terminus). Meteredamounts of material are then dispensed from the left arm into the rightarm of the horizontal channel by switching the applied voltage gradientfrom left to top, to left to right. The amount of time and the voltagegradient applied dictates the amount of material that will be dispensedin this manner. Although described for the purposes of illustration withrespect to a four way, cross intersection, these controlledelectrokinetic material transport systems can be readily adapted formore complex interconnected channel networks, e.g., arrays ofinterconnected parallel channels.

[0119] An example of a system employing this type of electrokinetictransport system in a microfluidic device, e.g., as illustrated in FIG.7, is shown in FIG. 11. As shown, the system 1100 includes amicrofluidic device 700, which incorporates an integratedpipettor/capillary element 708. Each of the electrical access reservoirs706, has a separate electrode 1128-1136 disposed therein, e.g.,contacting the fluid in the reservoirs. Each of the electrodes 1128-1136is operably coupled to an electrical controller 508 that is capable ofdelivering multiple different voltages and/or currents through thevarious electrodes. Additional electrode 1138, also operably coupled tocontroller 1108, is positioned so as to be placed in electrical contactwith the material that is to be sampled, e.g., in multiwell plate 502,when the capillary element 708 is dipped into the material. For example,electrode 1138 may be an electrically conductive coating applied overcapillary 708 and connected to an electrical lead which is operablycoupled to controller 508. Alternatively, electrode 1138 may simplyinclude an electrode wire positioned adjacent the capillary so that itwill be immersed in/contacted with the sample material along with theend of the capillary element 708. Alternatively, the electrode may beassociated with the source of material, as a conductive coating on thematerial source well or as a conductive material from which the sourcewell was fabricated. Establishing an electric field then simply requirescontacting the electrical lead with the source well material or coating.Additional materials are sampled from different wells on the multiwellplate 502, by moving one or more of the plate 502 and/or device 700relative to each other prior to immersing the pipettor 1138 into a well.Such movement is typically accomplished by placing one or more of thedevice 700 or multiwell plate 502 on a translation stage, e.g., theschematically illustrated x-y-z translation stage 1142.

[0120] In still a further optional application, hybrid materialtransport methods and systems may be employed. Briefly, one embodimentof such hybrid systems relies upon the use of electrokinetic forces togenerate pressure differentials within microfluidic systems. Such hybridsystems combine the controllability of electrokinetic systems with theadvantages of pressure based systems, e.g., lack of electrophoreticbiasing effects. Such hybrid systems are described in, e.g., PublishedInternational Patent Application No. WO 99/16162, which is incorporatedherein by reference in its entirety for all purposes. Other hybridsystems optionally employ electrokinetic forces to move materials in oneportion of the channel network, while employing pressure based forces inother portions of the channel network.

[0121] A variety of other systems may be employed in practicing thepresent invention including without limitation, e.g., rotor systems,dipstick systems, spotted array systems and the like.

IV. KITS AND REAGENTS

[0122] The reagents for carrying out the methods and assays of thepresent invention are optionally provided in a kit form to facilitatethe application of these assays for the user. Such kits also typicallyinclude instructions for carrying out the subject assay, and mayoptionally include the fluid receptacle, e.g., the cuvette, multiwellplate, microfluidic device, etc. in which the reaction is to be carriedout.

[0123] Typically, reagents included within the kit include the firstreagent that bears the fluorescent label, as well as the polyioniccompound. These reagents may be provided in vials for measuring by theuser, or in pre-measured vials or ampoules which are simply combined toyield an appropriate reaction mixture. The reagents may be provided inliquid and/or lyophilized form and may optionally include appropriatebuffer solutions for dilution and/or rehydration of the reagents.Typically, all of the reagents and instructions are co-packaged in asingle box, pouch or the like that is ready for use.

V. EXAMPLES Example 1 Detection of Phosphorylated Product by FluorescentPolarization

[0124] An aliquot of a neutrally charged phosphorylatable substrate(Flourescein-QSPKKG-CONH₂) (SEQ. ID NO. 12) was incubated overnight withATP and CDK2 (cyclin dependent kinase). The mixture was analyzed bystandard capillary electrophoresis methods and showed completeconversion of substrate to product. A negative control (no enzyme) wasalso prepared. The two reaction mixtures were diluted in 50 mM TAPS pH9.0 buffer (1:40). The fluorescence polarization values were measured byexciting the samples at 490 nm and measuring emitted fluorescence at 520nm in a cuvette of a fluorimeter equipped to measure fluorescencepolarization. Aliquots of a poly-D-Lysine solution and water were added(each added aliquot increased the poly-D-Lysine concentration by 6 μM).The results of the assay are illustrated in FIG. 12A which plots thefluorescent polarization of the sample versus the amount ofpoly-D-lysine added.

[0125] As shown, the fluorescence polarization of both substrate(square) and product (diamond) in the absence of polylysine was about 38milli polarization units (mP). Upon addition of polylysine, thefluorescence polarization of the product increased significantly (to 72and then to ˜100 mP upon addition of a large excess of polylysine). Thefluorescence polarization of the substrate only increased to about 42mP.

[0126] FIGS. 12B-12E are plots of fluorescent polarization of differentProtein Kinase A (PKA) and Protein Kinase C (PKC) substrates in thepresence of increasing concentrations of polyarginine. Specifically, anumber of PKC substrates and their phosphorylated derivatives wereanalyzed for fluorescence polarization in the presence of increasingconcentrations of polyarginine. The following substrates and theirphosphorylated derivatives were used at concentrations of 125 nM. Thenonphosphorylated peptides are represented in each of FIGS. 12B-12E bysquares, whereas the phosphorylated peptides are represented bydiamonds. In every case, the phosphorylated substrate yields a higherlevel of polarization. The peptides used are as follows: Substrate(Phos. Res. Charge Underlined) SEQ. ID NO. Enzyme (pH 7.5) FIG.F1-LRRASLG-CONH₂ 13 PKA 0 12B F1-LRRSSLG-COO⁻ 14 PKA −1 12CF1-KRPSKRAKA-COO⁻ 15 PKC 2 12D F1-KRTLRR-COO⁻ 16 PKC 1 12E

Example 2 Differentiation of Product Concentrations Using FluorescencePolarization

[0127] Additional experiments were carried out using poly-histidine inplace of polylysine. In this case, the buffer used was 50 mM BisTris pH6.5; the molecular weight of the polyhistidine used was 15800 daltons(available from Sigma Chemical, St. Louis, Mo.).

[0128] Mixtures containing varying ratios of the substrates and productsof two serine/threonine kinases were prepared, CDK2 and Protein Kinase A(PKA). The CDK substrate was the same as that described for Example 1,above. The PKA substrate was: Fluor-LRRASLG (SEQ. ID NO. 14) where theC-terminus was either a carboxyl group or a carboxamide group. Thesemixtures were used as models for kinase reactions at varying degrees ofsubstrate conversion. To these mixtures of substrate and product wereadded aliquots of a polyhistidine solution and water. The concentrationof this aqueous stock was approximately 1.3 mM, and the finalconcentration was between 10 and 25 mM.

[0129] Fluorescence polarization readings were again obtained byexciting at 490 and detecting emitted fluorescence at 520 nm (bothsubstrates were fluorescein labeled). FIGS. 13 and 14 show the resultsfrom these experiments. Briefly, FIGS. 13 and 14 are plots offluorescence polarization in increasing concentrations of phosphorylatedproduct as compared to substrate (denoted % conversion). In the case ofFIG. 13, the substrate and product are model substrate/products of aCDK2, while FIG. 14 illustrates similar data for PKA substrate/productmixtures (e.g., as described above). As can be seen, a very good lineardependence is observed between the fluorescence polarization signal andthe percent conversion of substrate to product. Thus, the method is wellsuited to follow the progress of kinase reactions and also for thescreening of chemical libraries for kinase inhibitors.

[0130] Protein Kinase A (PKA) and additional protein kinases were testedin similar assay procedures, but using substrates tailored for eachkinase, and using polyarginine as the polyion component. In particular,five different fluorescein-labeled peptides were prepared, containingsubstrate recognition sequences for three different serine or tyrosinekinase enzymes. The different substrates, in their unphosphorylatedstate, carried net charges of from +2 to −1 at pH 7.5. These chargeswould therefore yield phosphorylated products having charges from 0 to−3, respectively.

[0131] The various peptides were treated with their respective kinasesin the presence of ATP, and the amount of conversion was determinedusing capillary electrophoretic separation of substrate and product, aswell as by fluorescence polarization. All samples showed goodcorrelation between the CE and FP detection methods. As an example, thecorrelation between CE and FP detection for PKBα is shown in FIG. 15.

Example 3 Time Course Monitoring of Enzyme Reactions by FluorescencePolarization

[0132] Another PKA assay was performed with varying concentrations ofATP (0 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM and 32 μM) in 50 mMHEPES, pH 7.5, 10 mM MgCl₂, 500 nM polyarginine, 184 nM PKA, and 125 nMKemptide substrate (Fl-LRRASLG-COO⁻) (SEQ. ID NO. 14). The resultingassays were monitored over time in order to determine the efficacy ofthe fluorescence polarization detection methods of the present inventionon monitoring reaction time courses. FIG. 16 is a plot of fluorescencepolarization vs. reaction time for each different concentration of ATPin the reaction mix. As can be seen, increasing concentrations of ATPgenerally give faster reaction rates. In all cases except the control,fluorescence polarization measurements increase with time. Inparticular, as the reactions progress, more of the fluorescent substrateis rendered charged by virtue of the added phosphate groups, allowinggreater binding of polyarginine, and its associated changes influorescence polarization (e.g., more polarized, less depolarized). FIG.17 illustrates a plot of initial rate vs. ATP concentration, whichyields a characteristic kinetic plot for the assayed reaction. Thiskinetic data was then used in a Lineweaver-Burke plot (FIG. 18) todetermine the Km of the particular kinase enzyme.

Example 4 Assaying Phosphatase Activity by Fluorescence Polarization

[0133] The fluorescent polarization detection methods of the presentinvention were also applied in monitoring the time course of aphosphatase assay. Briefly, a fluorescent substrate for a knownphosphatase enzyme was placed in an assay buffer of 50 mM HEPES, pH 7.5,5 mM DTT, 200 mM NaCl, and 300 nM polyarginine. The relativefluorescence polarization level was monitored over time for a controlreaction (no enzyme) and a reaction mixture with differentconcentrations of phosphatase enzyme. FIG. 19 illustrates the plot forthe control and enzyme assay mixtures. As can be seen, the relativefluorescence polarization measurements decrease over time in thepresence of phosphatase enzyme. In particular, the mixture yields lesspolarized fluorescence over time as a result of less of the polyarginineinteracting with the fluorescent substrate due to the removal of thecharged phosphate group on the substrate which facilitates polyargininebinding.

Example 5 Assaying Protease Activity by Fluorescence Polarization

[0134] The fluorescent polarization detection methods of the presentinvention were also demonstrated for protease assays. In particular, achymotrypsin assay was carried out using these methods, using afluorescent, neutrally charged chymotrypsin specific substrate(Fl-EGIYGVLFKKK-CONH₂) (SEQ. ID NO. 17) bearing a fluorescent group atone end and a polylysine tail at the other. Specifically, cleavage ofthe above substrate by chymotrypsin yields Fl-EGIY (SEQ. ID NO. 18)having a net charge of −4 and GVLFKKK (SEQ. ID NO. 19) having a netcharge of +4. As described herein, a polycation will preferentiallyassociate with the highly negatively charged, fluorescent portion of thereaction products, yielding a shift in fluorescence polarization of thatlabeled portion. Briefly, the assay was carried out in 50 mM HEPESbuffer at pH 7.5, 5 mM CaCl₂, 500 nM chymotrypsin substrate, 1 μMpolyarginine. Three separate assays were run, a control run having noenzyme, and two assay runs having different concentrations of enzyme(0.125 μg/ml and 1.25 μg/ml chymotrypsin). FIG. 20 illustrates a plot offluorescent polarization versus time for each of the different assayruns. As shown, the control run (diamonds) showed no increase in thelevel of polarized fluorescence over time, while the low chymotrypsinconcentration (0.125 μm/ml, big squares, middle line) and higherchymotrypsin concentration (1.25 μg/ml, small squares, top line) showedincreasing levels of polarization over time, with the higher enzymeconcentration showing a faster initial increase. Increasing levels ofpolarized fluorescence is indicative of higher amounts of interaction ofpolyarginine with the negatively charged fluorescent portion of thesubstrate once the positively charged terminus is removed bychymotrypsin cleavage.

Example 6 Nucleic Acid Hybridization Assay Using FluorescencePolarization Detection

[0135] The assay methods were also employed in the detection of anucleic acid hybridization reaction. This assay is particularlyinteresting due to the lack of an immobilized target sequence that wasto be interrogated. In particular, the entire assay was carried out insolution.

[0136] A fluorescein-labeled peptide nucleic acid molecule 202 was usedin hybridization experiments with the DNA targets 192 and 182. PNAs aregenerally commercially available from the Applied Biosystems Division ofthe Perkin-Elmer Corporation (Foster City, Calif.). The sequence ofthese molecules is illustrated below. In the case of the PNA molecule,the given sequence illustrates the analogous sequence of a DNA molecule.The sequences of the three molecules are as follows: 202:5′Fl-O-GTCAAATACTCCA (SEQ.ID NO:1) 192: 5′ATGGGCTGGAGTATTTGACCTAATT(SEQ.ID NO:2) 182: 5′CGCTGTGGATGCTGCCTGA (SEQ.ID NO:3)

[0137] DNA sequence 192 contains 13 bases (shown in bold) that are fullycomplementary to the PNA probe 202, whereas 182 is a non-complementaryoligonucleotide.

[0138] A solution containing 1 μM of PNA 202 in 50 mM HEPES pH 7.5 wasmixed with either 5 μM of 192 or 5 μM of 182. The mixtures were left atroom temperature for about 10 min. and then placed in the cuvette of afluorimeter equipped to measure fluorescence polarization. Themeasurements were carried out using 490 nm for excitation and 520 nm fordetection of fluorescence emission. The fluorescence polarization valueswere recorded first in the absence and then in the presence ofpoly-L-Lysine hydrobromide, having a molecular weight of approximately70-100 kD (Sigma Chemicals). The poly-L-Lysine was added from a stocksolution in water (approx. 440 μM). The final concentrations of the polyLysine were 4.4 and 8.8 μM. The resulting fluorescence polarizationvalues are shown in FIG. 21.

[0139] As can be seen, a polarization value of 86 mP was obtained forthe mixture containing 202 and the non-complementary 182. This increasedto 140 mP in the presence of poly-L-Lysine. In contrast, the 202 hybridwith the complementary 192 sequence showed a polarization value of about100 mP in the absence (not shown in FIG. 21) and 229 mP in the presenceof poly-L-Lysine. These results demonstrate that fluorescencepolarization in the presence of poly-L-Lysine can be used to detect theformation of specific PNA/DNA hybrids in solution. Such assays arereadily employed in detection of the presence or absence of a particularsequence within a sample, e.g., in sequencing by hybridization, sequencechecking, screening for sequence variants, e.g., polymorphisms, i.e.,SNPs, STRs, and the like.

Example 7 Detection of Single Nucleotide Substitution

[0140] The assay methods were employed to detect differentialhybridization of a nucleic acid probe to a perfectly complementarytarget sequence and a target sequence incorporating a single basevariation, e.g., a single nucleotide polymorphism (SNP). In particular,a fluoresceinated PNA probe having a sequence complementary to asubsequence of a target sequence was used to probe the target includingthe subsequence and a target in which an interior base of thesubsequence was substituted for a different base.

[0141] The 5′ to 3′ sequences of the PNA probe (PNA 7637)(SEQ ID NO: 4),the target DNA sequence (DNA 244)(SEQ ID NO: 5) and single base mismatchtarget DNA sequence (DNA 245)(SEQ ID NO: 6) were as follows: PNA 7637:Fluorescein-CCTGTAGCA (SEQ ID NO:4) DNA 244: TTGTTGCCAATGCTACAGGCATCGT(SEQ ID NO:5) DNA 245: TTGTTGCCAATGCTGCAGGCATCGT (SEQ ID NO:6)

[0142] The subsequence of the DNA targets complementary to the PNA probeare in bold. The position of the SNP is underlined.

[0143] The PNA probe 7637 was used at a final concentration of 250 nM,in a reaction volume of 400 μl. The buffer used was 50 mM HEPES pH 7.5,100 mM NaCl. The solution also contained poly-L-Lysine at about 3 μM.One μl aliquots of the DNA targets 244 and 245 were added iteratively tothe PNA solution and the fluorescence polarization (excitation 490,emission 520 nm) was recorded after the addition of each aliquot. Thedata from this experiment is illustrated in FIG. 22. The perfectlycomplementary target/probe mixture (diamonds) showed substantiallyhigher levels of fluorescence polarization, than the single basemismatched mixture (squares), indicating that a higher level ofhybridization had occurred. As can also be seen, hybridization plateauedat approximately a 1 M excess of target sequence in the perfectlycomplementary example.

[0144] Thermal denaturation experiments were also performed in thepresence of polylysine, while monitoring fluorescence polarizationchanges in the reaction mixtures. In particular, three different targetDNA molecules were used having the following sequences:

[0145] 212: GCTGGAGTATTTGACCT (Perfect Match, ♦) (SEQ ID NO: 7)

[0146] 214: GCTGGAGTTTTTGACCT (T/T Mismatch in the middle, ▪) (SEQ IDNO: 8)

[0147] 215: GCTGGAGTCTTTGACCT (C/T Mismatch in the middle, ▴) (SEQ IDNO: 9)

[0148] Each target was interrogated with each of three differentfluorosceinated PNA probes having the following sequences (9-mer, 11-merand 13-mer) and subjected to increasing temperatures while thefluorescence polarization values of the mixtures were monitored: 188Fl-O-CAAATACTC (SEQ ID NO:10) 201 Fl-O-TCAAATACTCC (SEQ ID NO:11) 202Fl-O-GTCAAATACTCCA (SEQ ID NO:1)

[0149] Each of the reaction mixtures was then subjected to increasingtemperatures while the fluorescence polarization level was monitored.FIGS. 23A, B and C show each target, respectively, interrogated witheach of the three different probes. Data points represent average datafrom three separate experiments. All of the melting curves arenormalized where the melting curves from the PNA probes, alone aresubtracted from the melting curves generated in the presence of the DNAtarget sequences.

[0150] As would be expected, the longer the PNA probe used, the fartherout the melting curve is pushed. In particular, FIG. 23A shows a muchlower melting point for the target and probe (9-mer) than FIGS. 23B(11-mer) and 23C (13-mer). Further, clear discrimination can be seenbetween the perfectly matched hybrids (diamonds) and the single basemismatched hybrids (squares and triangles) for each target sequence,with the C/T mismatch, among the two presented, being the mostdestabilizing, i.e., yielding the greatest shift in the melting curve.This example clearly illustrates the sensitivity with which thepresently described methods can be used to discriminate singlenucleotide differences between target sequences, e.g., SNPs, etc.

[0151] Although the above-described assays utilized fluorescencepolarization detection, it has also been discovered that these assaymethods yield changes in fluorescence intensity upon hybridization. Inparticular, single nucleotide substitution assays, like those describedabove, were run on two separate nucleic acid sequences. In each, a PNAprobe (250 nM) that is complementary to the wild type and one having asingle base substitution, were used to probe the target sequence (in 50mM HEPES, pH 7.5, 100 mM NaCl), followed by treatment with poly-L-lysine(3.3 μM). The mixtures were exposed to increasing concentrations of thetarget sequence, and the fluorescence polarization and totalfluorescence intensity were measured. FIG. 24 is a plot of bothfluorescent intensity and fluorescence polarization for each of theperfect hybrids and single base mismatches tested. As can be seen, bothfluorescent intensity and fluorescence polarization provide a basis fordistinguishing between the perfect match and single base mismatchreactions.

[0152] Unless otherwise specifically noted, all concentration valuesprovided herein refer to the concentration of a given component as thatcomponent was added to a mixture or solution independent of anyconversion, dissociation, reaction of that component to a alter thecomponent or transform that component into one or more different speciesonce added to the mixture or solution. The method steps described hereinare generally performable in any order unless an order is specificallyprovided or a required order is clear from the context of the recitedsteps. Typically, the recited orders of steps reflects one preferredorder.

[0153] All publications and patent applications are herein incorporatedby reference to the same extent as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference. Although the present invention has beendescribed in some detail by way of illustration and example for purposesof clarity and understanding, it will be apparent that certain changesand modifications may be practiced within the scope of the appendedclaims.

1 19 1 13 DNA Artificial Sequence PNA probe 1 gtcaaatact cca 13 2 25 DNAArtificial Sequence PNA probe 2 atgggctgga gtatttgacc taatt 25 3 19 DNAArtificial Sequence PNA probe 3 cgctgtggat gctgcctga 19 4 9 DNAArtificial Sequence PNA probe 4 cctgtagca 9 5 25 DNA Artificial SequencePNA probe 5 ttgttgccaa tgctacaggc atcgt 25 6 25 DNA Artificial SequencePNA probe 6 ttgttgccaa tgctgcaggc atcgt 25 7 17 DNA Artificial SequencePNA probe 7 gctggagtat ttgacct 17 8 17 DNA Artificial Sequence PNA probe8 gctggagttt ttgacct 17 9 17 DNA Artificial Sequence PNA probe 9gctggagtct ttgacct 17 10 9 DNA Artificial Sequence PNA probe 10caaatactc 9 11 11 DNA Artificial Sequence PNA probe 11 tcaaatactc c 1112 6 PRT Artificial Sequence Phosphorylatable substrate 12 Gln Ser ProLys Lys Xaa 1 5 13 7 PRT Artificial Sequence Phosphorylatable substrate13 Leu Arg Arg Ala Ser Leu Xaa 1 5 14 7 PRT Artificial SequencePhosphorylatable substrate 14 Leu Arg Arg Ala Ser Leu Gly 1 5 15 9 PRTArtificial Sequence Phosphorylatable substrate 15 Lys Arg Pro Ser LysArg Ala Lys Ala 1 5 16 6 PRT Artificial Sequence Phosphorylatablesubstrate 16 Lys Arg Thr Leu Arg Arg 1 5 17 11 PRT Artificial SequenceProtase substrate 17 Glu Gly Ile Tyr Gly Val Leu Phe Lys Lys Xaa 1 5 1018 4 PRT Artificial Sequence Protease product 18 Glu Gly Ile Tyr 1 19 7PRT Artificial Sequence Protease product 19 Gly Val Leu Phe Lys Lys Lys1 5

What is claimed is:
 1. A method of measuring the activity of a kinase enzyme, comprising: providing a reaction mixture comprising a fluorescently labeled phosphorylatable compound, a kinase enzyme and a phosphate donor group, wherein the kinase enzyme is capable of transferring a phosphate from the phosphate donor group to the phosphorylatable compound to produce a phosphorylated product; contacting the phosphorylated product with a molecule having multivalent metal cations associated therewith; and determining a level of phosphorylated product by detecting a level of fluorescent intensity emitted from the reaction mixture.
 2. The method of claim 1, wherein the compound comprises a serine, tyrosine, or threonine substrate.
 3. The method of claim 1, wherein the multivalent metal cations bind the molecule to the phosphorylated product at least partially because of a difference in charge between the phosphorylated product and the multivalent metal cations.
 4. The method of claim 1, wherein the multivalent metal cations bind the molecule to the phosphorylated product at least partially because of a specific binding affinity between the metal cations and a phosphate group associated with the phosphorylated product.
 5. The method of claim 1, wherein the multivalent metal cations comprise trivalent metal cations.
 6. The method of claim 5 wherein the trivalent metal cations comprise Fe³⁺.
 7. The method of claim 1, further comprising introducing at least a first test compound into the reaction mixture and comparing the level of fluorescent intensity emitted from the reaction mixture in the presence of the test compound to the level of fluorescent intensity emitted from the reaction mixture in the absence of the test compound.
 8. The method of claim 7, further comprising repeating the providing, introducing and comparing steps with a plurality of different test compounds.
 9. The method of claim 1, wherein the molecule comprises a polymer.
 10. A method of measuring the activity of a phosphatase enzyme, comprising: providing a reaction mixture comprising a fluorescently labeled phosphorylated compound, a phosphatase enzyme, and a molecule having multivalent metal cations associated therewith; and determining a level of dephosphorylated product produced by the activity of the phosphatase enzyme by detecting a level of fluorescent intensity emitted from the reaction mixture.
 11. The method of claim 10, wherein the fluorescent intensity increases in proportion to the amount of dephosphorylated product in the reaction mixture.
 12. The method of claim 10, wherein the multivalent metal cations comprise trivalent metal cations.
 13. The method of claim 12, wherein the trivalent metal cations comprise Fe³⁺.
 14. The method of claim 10, further comprising introducing at least a first test compound into the reaction mixture and comparing the level of fluorescent intensity emitted from the reaction mixture in the presence of the test compound to the level of fluorescent intensity emitted from the reaction mixture in the absence of the test compound.
 15. The method of claim 14, further comprising repeating the providing and comparing steps with a plurality of different test compounds.
 16. The method of claim 10, wherein the molecule comprises a polymer.
 17. A method of monitoring the activity of an enzyme, comprising: providing a first mixture comprising a fluorescently labeled substrate and an enzyme, wherein the enzyme is capable of modifying the chemical structure of the substrate to produce a fluorescently labeled product; contacting the product with a molecule having multivalent metal cations associated therewith; and determining a level of product produced by the activity of the enzyme by measuring binding of the molecule to the product.
 18. The method of claim 17, wherein the enzyme is capable of modifying the chemical structure of the substrate by addition to, subtraction from, or alteration of its chemical structure.
 19. The method of claim 17, wherein the substrate comprises a serine, tyrosine, or threonine substrate.
 20. The method of claim 17, wherein the multivalent metal cations bind the molecule to the product based at least partially on a difference in charge between the product and the multivalent metal cations.
 21. The method of claim 17, wherein the multivalent metal cations bind the molecule to the product based at least partially on an affinity between the metal cations and a phosphate group associated with the product
 22. The method of claim 17, wherein the multivalent metal cations comprise trivalent metal cations.
 23. The method of claim 22, wherein the trivalent metal cations comprise Fe³⁺.
 24. The method of claim 17, wherein the substrate compriss a phosphorylated substrate and the enzyme comprises a phosphatase enzyme.
 25. The method of claim 17, wherein the substrate comprises an amino or keto containing substrate and the enzyme comprises an amino transferase.
 26. The method of claim 17, wherein the substrate includes a substrate for one of the following: a sulfatase, a phosphorylase, an esterase, a hydrolase, an oxidase, or an analog thereof.
 27. The method of claim 17, wherein the determining step is performed using fluorescence polarization detection.
 28. The method of claim 17, wherein the determining step is performed using fluorescence intensity detection. 